Proline-directed phosphorylation (pSer/Thr-Pro) is a major mechanism for controlling cell growth and transformation. Although phosphorylation is thought to regulate protein function by inducing conformational changes, little is known about most of these conformational changes and their importance. Recent identification of the prolyl isomerase Pin1 that specifically isomerizes only pSer/Thr-Pro bonds in certain proteins led to a proposal of a new signaling mechanism, whereby prolyl isomerization catalytically induces conformational changes in proteins following phosphorylation to regulate their function. Emerging data indicate that such conformational changes can have profound effects on protein function. We also have found that Pin1 is overexpressed in many cancers and positively regulates cyclin D1 at the transcriptional level and through post-translational stabilization. Pin1 knockout mice display many phenotypes resembling cyclin D1- null phenotypes. Thus, Pin1 is an important new regulator of cyclin D1, a known key mediator in oncogenesis. However, it remains to be determined how Pin1 function itself is regulated, and whether and how Pin1 plays a role in tumorigenesis. This proposal represents our continuous effort to understand Pin1 function and regulation especially during oncogenesis. Pin1 phosphorylation is regulated during the cell cycle and altered in cancer cells. Pin1 mRNA also is elevated in cancer tissues and some oncogenes might activate the Pin1 promoter. Aim 1 and Aim 2 are therefore designed to investigate how Pin1 function is regulated by phosphorylation and transcriptional control, respectively. Importantly, Pin1 overexpression might confer some transformed phenotypes to normal cells and also enhance the transformed phenotype of some oncogenes, whereas Pin1 inhibition induces apoptosis in cancer cells and might also suppress the transformed phenotypes induced by Ras and Neu. Aim 3 and 4 are therefore designed to address whether and how overexpression and inhibition of Pin1 play any significant roles in the development and suppression of oncogenesis, respectively, using in vitro and in vivo models. These studies should help elucidate how Pro-directed phosphorylation regulates the cell cycle and tumorigenesis, but may also have novel implications for cancer therapy.